Method for the amplification of nucleic acids using heat transfer for nanoparticles

ABSTRACT

A method for the amplification of nucleic acids, in which nanoparticles in a reaction volume transfer heat to their environment through excitation. The method comprises a step of providing nanoparticles with the nucleic acids in a reaction volume and one or more heating steps. In at least one of the heating steps, the heating is achieved at least partially through the excitation of the nanoparticles. The interval of the excitation is chose to be shorter or equal to a critical excitation time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/375,946, filed Jul. 31, 2014, which is a National Phase ofPCT/EP2013/052100, filed Feb. 1, 2013, which claims priority to Germanpatent application no. DE 102012201475.6, filed Feb. 1, 2012, thedisclosures of each of which are incorporated herein by reference.

FIELD OF THE DISCLOSURE

The invention concerns a method for the amplification of nucleic acids.

BACKGROUND OF THE INVENTION

Methods for the amplification of nucleic acids are known in the art. Thepatent specification U.S. Pat. No. 4,683,202 discloses a process foramplifying a specific nucleic acid sequence contained in a nucleic acidor a mixture of nucleic acids, wherein each nucleic acid consists of twoseparate complementary strands, of equal or unequal length. The processcomprises: (a) treating the strands with two oligonucleotide primers,for each different specific sequence being amplified, under conditionssuch that for each different sequence being amplified an extensionproduct of each primer is synthesised which is complementary to eachnucleic acid strand, wherein said primers are selected so as to besufficiently complementary to different strands of each specificsequence such that the extension product synthesised from one primer,when it is separated from its complement, can serve as a template forsynthesis of the extension product of the other primer; (b) separatingthe primer extension products from the templates on which they weresynthesised to produce single-stranded molecules; and (c) treating thesingle-stranded molecules generated from step (b) with the primers ofstep (a) under conditions that a primer extension product is synthesisedusing each of the single strands produced in step (b) as a template. Thesteps can be carried out consecutively or simultaneously. Furthermore,the steps (b) and (c) can be repeated until the desired extent ofsequence amplification has been achieved. In the case that in theprocess the steps (a) and (c) are performed using a polymerase, theprocess is commonly referred to as polymerase chain reaction (PCR).

The international patent application WO 2007/143034 A1 disclosesmethods, which are supposedly suitable for the execution of PCR. Themethods may comprise the use of an optical source to provide heating ina PCR. The methods may also include the use of surface plasmon resonanceor fluorescence resonance energy transfer to allow real-time monitoringof a PCR reaction. The methods may comprise immobilising a template,primer or polymerase on a surface such as a gold or on another surfaceplasmon resonance active surface.

The patent application US 2002/0061588 A1 discloses methods forrendering nucleic acids locally and directly responsive to an externalsignal. The signal acts exclusively on one or several specific,localised parts of the nucleic acid. According to the invention, thesignal can change the properties of a specific nucleic acid and therebymodify its function. Thus, the invention provides methods, which controlthe structure and function of a nucleic acid in a biological samplewithout influencing other parts of the sample. In one embodiment, amodulator transfers heat to a nucleic acid or a part of a nucleic acid,which results, e.g., in a destabilisation of inter- or intramolecularbonds and in an alteration of structure and stability of the nucleicacid. Preferred modulators include metal nanoparticles, semiconductingnanoparticles, magnetic nanoparticles, oxide nanoparticles andchromophores. It is also suggested, to use these methods in conjunctionwith PCR. Particularly, it is proposed to control a PCR reaction with amodulator.

The patent application US 2003/0143604 A1 concerns the use ofnanoparticle detection probes to monitor amplification reactions, inparticular PCR. Especially, the patent application deals with the use ofnanoparticle oligonucleotide conjugates treated with a protective agentsuch as bovine serum albumin in order to quantitatively andqualitatively detect a target polynucleotide. The patent applicationdiscloses a nucleic acid amplification and detection using goldnanoparticle primers. In a first step, the nucleic acid target isdenatured in the presence of the gold nanoparticles that arefunctionalised with primers. In a second step, the gold nanoparticlesand the oligonucleotides attached thereto hybridise with the nucleicacid target and a copy of the complementary DNA sequence is producedstarting from the nucleic acid primers, which are attached to thenanoparticles. The steps one and two are repeated and the opticalsignal, which is created by the binding of amplified complementarynanoparticle probes, is detected.

Problem According to the Invention

The underlying problem of the invention is to provide an improved methodfor the amplification of nucleic acids.

Solution According to the Invention

The problem is solved by a method with the features of claim 1. Themethod serves to amplify nucleic acids, wherein in a reaction volume,nanoparticles transfer heat to their environment through excitation.

The reaction volume is the volume, in which the method according to theinvention is performed. The volume can be surrounded by a reactionvessel. The reaction volume contains a sample. The sample contains aliquid, preferably water. The nucleic acids, which can be amplified bythe method, can be contained in the sample.

The nanoparticles according to the invention are preferably particles,which, due to their size, show special optical properties, particularlycharacteristic absorption or scattering spectra, that are not observedor not as distinct in the bulk material. Preferably, the nanoparticleshave a diameter of between 2 and 500 nm, more preferably between 3 and300 nm and most preferably between 5 and 200 nm. Preferred nanoparticleshave a diameter between 7 and 150 nm. The nanoparticles can bespherical, however, non-globular shapes are also possible, e.g.,elongated nanoparticles (nanorods). In a preferred embodiment of theinvention, the nanoparticle comprises at least one semiconductor or onemetal, preferably a noble metal, e.g., gold or silver. In oneembodiment, the nanoparticle consists of metal entirely, in anotherembodiment the metal forms only one part of the nanoparticle, e.g., itsshell. A preferred nanoparticle can be a shell-core nanoparticle. Apreferred nanoparticle can possess pores on its surface, which pores canbe occupied by atoms or molecules with a size and charge defined by theproperties of the pores, particularly preferably, such atoms ormolecules are only adsorbed to the nanoparticle when the nanoparticle issituated in a solution. According to the invention, the nanoparticlealso comprises the atoms and molecules adsorbed to its surface. Due totheir material absorption or plasmon resonance, preferred nanoparticlesare suitable for absorbing optical energy.

When—through the excitation of a nanoparticle—heat is transferred fromthe nanoparticle to its environment, this means—according to theinvention—that energy is transferred to the nanoparticle, wherein thenanoparticle heats its environment through the transfer of the energy.In this, preferentially, through the excitation, the immediateenvironment is heated more strongly than the wider environment of thenanoparticles. Typically, the nanoparticles are first heated throughexcitation and then transfer heat to their environment. It is alsoconceivable that through the excitation of the nanoparticles, heat istransferred to their environment without the nanoparticles themselvesbeing heated first. Preferably, the environment of the nanoparticles isa spherical volume, which has a diameter equal to 100 times the diameterof the nanoparticle which is situated in the centre of the volume; morepreferably the volume has 10 times, most preferably 4 times andpreferably less than 2 times the diameter of the nanoparticle in itscentre.

Preferably, through the excitation of the nanoparticles, the environmentof the nanoparticles is heated locally. Especially fast changes intemperature are possible if the heated volume is only a fraction of theentire volume. On the one hand, a high temperature difference can beproduced with only a small amount of energy input. On the other hand, arapid cooling of the heated volume is possible if a sufficiently largecold temperature reservoir is present in the irradiated volume, suchthat after the irradiation of the nanoparticles, their environment iscooled down. This can be achieved by irradiating the nanoparticlessufficiently strongly (to gain the desired temperature increase) and fora sufficiently short amount of time (for the heat to remain localised).

Local heating according to the invention is thus present if the intervalt of the excitation in the individual volume irradiated (e.g., in thefocus of the laser) is chosen to be shorter or equal to a criticalexcitation interval t1. Here, t1 is the time which the heat requires todiffuse from one nanoparticle to the next at a mean nanoparticledistance, multiplied with a scaling factor s1; if |x| is the meannanoparticle distance and the thermal diffusivity of the medium betweenthe nanoparticles is D then t1 is given by t1=(s1*|x|)²/D, wherein thethermal diffusivity D typically has a value of D=10⁻⁷ m²/s in aqueoussolution.

The scaling factor s1 is a measure for how far the warm front of aparticle spreads during the excitation interval. The temperatureincrease caused by an excited nanoparticle at a distance of a fewnanoparticle diameters is only a very small fraction of the maximaltemperature increase at the particle surface. In one embodiment of theinvention, an overlap of the warm fronts of a few nanoparticles ispermitted in the sense that for the definition of the criticalexcitation interval t1 according to the equation above, a scaling factorgreater than 1 is used. In another embodiment of the invention, nooverlap of the warm fronts is permitted during the excitation interval(corresponds to a markedly local heating) in the sense that for thedefinition of the critical excitation interval t1 according to the aboveequation, a scaling factor s1 less than or equal to 1 is used. For thedefinition of the local heating according to the invention, preferablys1=100, preferably s1=30, preferably s1=10, preferably s1=7, preferablys1=3 and most preferably s1=1, preferably, s1=0.7, preferably s1=0.3.

Values for s1>1 can be advantageous in such cases (amongst others), inwhich the irradiated volume shows a high aspect ratio (e.g., in thefocus of a moderately focussed laser beam), such that comparatively manynanoparticles are situated at the surface of the irradiated volume andtherefore, fewer heated nanoparticles are present in their environmentand a marked heat efflux from the irradiated volume takes place, suchthat the heating contribution of the neighbours further away remainsnegligible for a longer period of time.

This means that, e.g., at a nanoparticle concentration of 1 nM, whichresults in a mean nanoparticle distance of |x|=1.2 μm, a local heatingaccording to the invention takes place if the excitation intervalremains shorter than t1=14 μs (the scaling factor is chosen as s1=1,D=10⁻⁷ m²/s). It can be assumed that when t is chosen to be t>t1 thatthe heat given off by the nanoparticles can—during irradiation—cover adistance by diffusion, which is greater than the mean particle distance,which in effect leads to an overlap of the warm fronts of manynanoparticles, such that there is a temperature increase in the entirevolume between the nanoparticles; the temperature increase in theirradiated volume will be spatially the more homogeneous the longer theheating takes place, as an influence on the temperature distributionaround a nanoparticle is not only exerted by the closest nanoparticles,but also by the neighbours further away.

If the reaction volume is irradiated with a radiation absorbed by thenanoparticles for longer than t1, the heating is termed as global.

A global heating according to the invention can, e.g., be carried out byheating the reaction volume from the outside with a Peltier element or aresistance heater. The global heating can also take place, e.g., byirradiating the reaction volume with radiation, which is absorbed bywater more strongly than or equally strongly as by the nanoparticles.Here, the term temperature increase means the difference in thetemperature at one location at the time of observation immediately afterthe excitation and the temperature at the same location immediatelybefore the excitation.

Global heating and local heating can be carried out simultaneously.

Known methods for the amplification of nucleic acids comprise one orseveral steps, in which at least parts of the sample are required to beheated.

The invention makes it achievable that in the method for theamplification of nucleic acids, not the entire reaction volume needs tobe heated. On the contrary, it is possible to only heat specific partsof the reaction volume through the excitation of nanoparticles.Advantageously, in such way, it becomes possible to only heat thoseparts of the reaction volume, which need to be heated for theamplification of the nucleic acids. Thus, heat sensitive parts of thesample can be preserved. The local heating can be faster than the globalheating of the entire reaction volume, if less energy needs to betransferred. Therefore, advantageously, the invention makes it possibleto provide a method for the amplification of nucleic acids, which isfaster and requires less energy.

Preferred Embodiments According to the Invention

A nucleic acid can be amplified by a polymerase chain reaction (PCR), inparticular. The PCR is carried out in the reaction volume. The reactionvolume contains one nucleic acid to be amplified, which is termed theoriginal. The original is a single strand. In the reaction volume, theoriginal can form a double strand together with its complementarystrand, which is termed the complement. If the original and thecomplement are present as a double strand, this double strand must bedenatured in a first step, i.e., the double strand must be split intotwo single strands. Melting is another term for denaturing. Denaturingoccurs at a temperature, which is termed denaturing temperature. Thereaction volume further contains at least two oligonucleotides, whichare called primers. One of the primers is termed forward primer, theother is called reverse primer. The forward primer is complementary tothe 3′-end of the original. The reverse primer is complementary to the3′-end of the complement. In a second step, the forward primerhybridises with the original and the reverse primer hybridises with thecomplement. The hybridisation of the primers with the complementaryparts of the original or the complement, respectively, is termedannealing. The second step takes place at a temperature, which is termedannealing temperature. The reaction volume further contains a DNApolymerase. In a third step, the DNA polymerase synthesises a copy ofthe complement starting from the forward primer. Starting from thereverse primer, the DNA polymerase synthesises a copy of the original.Through the synthesis, the copy of the complement is hybridised with theoriginal and the copy of the original is hybridised with the complement.The third step is termed elongation and is carried out at a temperaturecalled elongation temperature. After that, the first, second and thirdstep are cyclically repeated until the desired extent of amplificationis achieved, wherein the copy of the original is the original and thecopy of the complement is the complement. If the original is situated ona DNA single strand that is longer than the original, then the PCR doesnot only produce copies of the original, but also copies of the said DNAsingle strand, which are longer in the 3′ direction and contain theoriginal. Accordingly, in this case, the PCR does not only producecopies of the complement, but also copies of the DNA single strandcomplementary to the said DNA single strand, wherein the copies arelonger in the 3′ direction and contain the complement.

As the separate steps of the PCR can be carried out at differenttemperature, it can be necessary to perform one or several heating stepsand—where applicable—cooling steps during or between the steps of thePCR, in which heating and cooling steps the reaction volume or partsthereof are heated or cooled, respectively. Preferably, the heating inthe heating step or in at least one of the heating steps is achieved atleast partially through the excitation of the nanoparticles and theheating is preferably a local heating.

In the PCR, the denaturing temperature is preferably chosen such thatthe single strands of the DNA melt while not damaging the DNA polymerasein a significant way. A typical value for the denaturing temperature is,e.g., 95° C. The optimal annealing temperature usually depends on thesequence and length of the primers. Typically, primers are designed forthe annealing temperature to be between 50° C. and 65° C. The optimalelongation temperature typically depends on the DNA polymerase used.When using Taq polymerase, e.g., typically, an elongation temperature of72° C. is chosen.

Hybridisation in the sense of the present invention means the forming ofa double strand from two single strands, each of which may consist of anucleic acid and/or oligonucleotide. Under appropriate reactionconditions, the hybridisation typically leads to the lowest energystate, which can be achieved by the two single strands bonding to eachother. This means, in other words, that under the appropriateconditions, the two single strands bind to each other in such a way thatreferring to the sequences of the two single strands, the greatestpossible complementarity is produced.

When a nucleic acid A is partially complementary to a nucleic acid B,this means that one part of the nucleic acid A is complementary to onepart of the nucleic acid B.

The excitation of the nanoparticles preferably takes place by means ofan alternating field, more preferably by an alternating electromagneticfield, most preferably optically. Preferably, the excitation occurs inthe range between far infrared and far ultraviolet light (in a rangefrom 100 nm to 30 μm wavelength), more preferably in the range from nearinfrared to near ultraviolet light (in a range from 200 nm to 3 μmwavelength), most preferably in the visible light range (in a range of400 nm to 800 nm). Compared to the conventional global heating of thereaction vessel from the outside, this may offer the advantage that thethermically insulating wall of the reaction vessel does not need to beovercome as the energy is transferred directly onto the nanoparticles.In this way, a faster heating of the desired parts of the sample can beachieved.

In a preferred embodiment of the invention, the nanoparticles areexcited by a laser. More preferably, the laser light has a frequency,which excites the surface plasmon resonance of the nanoparticles. Thelaser can supply the light continuously or as pulsed light. The lasercan, e.g., be a gas laser, a diode laser or a diode-pumped solid statelaser. The time interval, in which the laser excites the nanoparticlesin the irradiated volume, is preferably in the area of picoseconds toseconds, more preferably between nanoseconds and seconds and mostpreferably between 10 ns and 500 μs. Preferably, the excitation intervalis shorter than the mean time needed for the heat, which arises in theenvironment of the nanoparticles, to diffuse across the meannanoparticle distance, such that there is, on average, no significantoverlap of the warm fronts of neighbouring particles. More preferably,the excitation interval is chosen for the temperature increase aroundeach irradiated nanoparticle to drop to, on average, less than half itsmaximum in a distance of 20 nanoparticle diameters, more preferably in adistance of 2 nanoparticle diameters and most preferably in a distanceof 1 nanoparticle diameter. In one embodiment, a short irradiationperiod of the laser per volume is preferable, such that a dehybridisedDNA single strand can only diffuse away from the nanoparticle by lessthan 100 nm, more preferably by less than 20 nm during the denaturation.Thereby, the probability for the dehybridised DNA single strand to bindto an oligonucleotide on the same nanoparticle is high. This can lead toan acceleration of the method according to the invention. In a preferredembodiment, the concentration of the primer conjugated nanoparticles issmaller than 10 nM, wherein the excitation interval is preferablybetween 1 ns and 10 μs, more preferably between 10 ns and 1 μs and mostpreferably between 15 ns and 300 ns. The excitation interval ispreferably not chosen to be significantly smaller than 1 ns; otherwisethe heating period of the DNA double strand is not sufficient for thesingle strands contained therein to sufficiently separate by diffusionsuch that they will not immediately rehybridise to each other.

The duty cycle is the ratio of the excitation interval to the durationof the PCR cycle. The duty cycle is preferably chosen to be large enoughfor the excitation to lead to a sufficient denaturation of the DNAdouble strands by local heating. At the same time, the duty cycle ischosen to be such that the mean temperature increase of the entiresample is kept sufficiently small to avoid disturbances on thehybridisation, elongation and denaturation. Preferably, the duty cyclefor the irradiated volume is less than 50%, more preferably less than20% and most preferably less than 1%. The duty cycle in the irradiatedvolume is preferably greater than 10⁻¹², more preferably greater than10⁻¹⁰, more preferably greater than 10⁻⁹ and most preferably greaterthan 10⁻⁸. The surface power densities, with which the nanoparticles areexcited, are preferably between 20 W/mm² and 1000 kW/mm², morepreferably between 100 W/mm² and 100 kW/mm² and most preferably between250 W/mm² and 10 kW/mm².

In another preferred embodiment, the energy of the laser light istransferred to the nanoparticles due to their material absorption. Thelight, which is used for the excitation of the nanoparticles, can alsooriginate from, e.g., a thermic radiator, e.g., a flash bulb. In anotherpreferred embodiment of the invention, the nanoparticles are excitedthrough an alternating electromagnetic field or electromagnetic waves,which induce eddy currents in the nanoparticles. Appropriately designednanoparticles can also be excited by ultrasound.

The term oligonucleotide in connection with the present inventionpreferably comprises not only (deoxy)oligoribonucleotides, but alsooligonucleotides that contain one or more nucleotide analogues withmodifications on their backbone (e.g. methylphosphonates,phosphothioates or peptide nucleic acids [PNA]), in particular on asugar of the backbone (e.g. 2′-O-alkyl derivatives, 3′- and/or5′-aminoriboses, locked nucleic acids [LNA], hexitol nucleic acids,Morpholinos, glycol nucleic acids (GNA), threose nucleic acid (TNA) ortricyclo-DNA; in this regard see the publication by D. Renneberg and C.J. Leumann entitled “Watson-Crick base-pairing properties ofTricyclo-DNA”, J. Am. Chem. Soc., 2002, Vol. 124, pages 5993-6002, therelated content of which forms part of the present disclosure by way ofreference) or contain base analogues, e.g., 7-deazapurine or universalbases such as nitroindole or modified natural bases such asN4-ethylcytosine. In one embodiment of the invention, theoligonucleotides are conjugates or chimeras with non-nucleosidicanalogues, e.g. PNA. In one embodiment, the oligonucleotides contain, atone or more positions, non-nucleosidic units such as spacers, e.g.hexaethyleneglycol or Cn-spacers, where n is between 3 and 6. To theextent that the oligonucleotides contain modifications, these are chosenin such a way that a hybridisation with natural DNA/RNA analytes is alsopossible with the modification. Preferred modifications influence themelting behaviour, preferably the melting temperature, in particular inorder to distinguish hybrids having differing degrees of complementarityof their amino acids (mismatch discrimination). Preferred modificationsinclude LNA, 8-aza-7-deaza-purine, 5-propinyluracil, 5-propinylcytosineand/or abasic interruptions in the oligonucleotide. Furthermodifications according to the invention are, e.g., modifications withbiotin, thiol and fluorescence donor and fluorescence acceptormolecules.

In a preferred embodiment of the invention, the nanoparticles areconjugated with oligonucleotides. In this way, the nanoparticles formnanoparticle oligonucleotide conjugates. In this manner, it can beachieved that oligonucleotides forming part of the method according tothe invention can be specifically heated through the excitation of thenanoparticles without having to heat the reaction volume as a whole. Inan especially preferred embodiment, the nanoparticles are conjugated toprimers. More preferably, the nanoparticles are conjugated to theforward and reverse primers of the PCR. In a preferred embodiment of theinvention, one kind of nanoparticle oligonucleotide conjugates haveforward primers but no reverse primers attached; another kind hasreverse but no forward primers attached.

In another preferred embodiment of the invention, one kind ofnanoparticle oligonucleotide conjugates is conjugated with forward aswell as reverse primers. In this embodiment, a new DNA single strandcomplementary to the original is synthesised in a PCR starting from theforward primer on a nanoparticle. This new DNA single strand isconjugated to the nanoparticle as the new DNA single strand contains theforward primer. Immediately after the synthesis, the new DNA singlestrand forms a double strand with the original. In a subsequentdenaturation step, the new DNA single strand is separated from theoriginal. At an annealing temperature, the new DNA single strandhybridises to a reverse primer, which is situated on the surface of thenanoparticle, such that a loop is formed. For the hybridisation with thereverse primer on the same nanoparticle, only a short distance has to becrossed. To achieve hybridisation with a reverse primer on a differentnanoparticle at preferred nanoparticle concentrations, on average, thedistance to be crossed is greater. Thus, in this embodiment, it isadvantageously achievable that the annealing occurs more rapidly andthat the PCR can be completed more quickly.

In a preferred embodiment of the invention, the nanoparticles areconnected to the primers in such a way that covalent bonds with morethan one thiol between primers and nanoparticles are present. Generally,PCR buffers contain dithiothreitol, which destabilises the thiol bondbetween the gold nanoparticles and the primers and which can—especiallyunder thermal strain, e.g., during denaturation—lead to primersdetaching themselves from the nanoparticles. Covalent bonds with morethan one thiol between the primers and the nanoparticles can decreasethe detachment of the primers and thus improve PCR efficiency.

In a preferred embodiment, countersequences are used, which can bind tosuch oligonucleotides, which have detached themselves from nanoparticleswith which they had been connected previously. Countersequences areoligonucleotides. In the method, it can occur that oligonucleotides,which are conjugated to nanoparticles, detach themselves from saidnanoparticles and become free. In the case that said freeoligonucleotides are the primers according to the invention, these freeprimers can bind to the original or the complement. As the free primersare not bound to the nanoparticles, the free primers cannot bedehybridised from the original or complement, respectively, byexcitation of the nanoparticles. Thereby, the efficiency and sensitivityof the method is decreased. The countersequences are at least partlycomplementary to the free oligonucleotides and bind to these withsufficient affinity to limit the function of the free oligonucleotides.In this way, the efficiency and sensitivity of the method can beincreased. In a particularly preferred embodiment of the method, anamount of countersequences sufficient to block free primers is added tothe sample even before the addition of the original. At the same time,said amount is small enough for the nanoparticles to display asufficiently large number of non-blocked primers. This is possible ifthe number of primers on the nanoparticles exceeds the number of freeprimers.

In a preferred embodiment, filling molecules are attached to thenanoparticles. The filling molecules prevent the undesired aggregationof the nanoparticles in the sample. Thus, the filling moleculesadvantageously serve to stabilise the nanoparticles. The charge of thenanoparticles can be modulated using the filling molecules. In this way,the salt concentration, which is present in the environment of thenanoparticles, can be adapted such that the DNA polymerase cansynthesise as quickly as possible and that, advantageously, the methodcan be performed rapidly. The filling molecules can consist ofoligonucleotides that are not primers and which are preferably shorterthan the primers. The filling molecules can also, e.g., consist ofpolymers, such as, e.g., polyethylene glycol. In a preferred embodiment,the filling molecules permit to decrease the number of primers on thenanoparticles and to instead use more filling sequences withoutdecreasing the efficiency of the method by a significant amount.

In a further preferred embodiment of the method, the oligonucleotides onthe nanoparticles show a spacer sequence as a partial sequence. Thespacer sequence is situated in the part of the oligonucleotide closer tothe nanoparticle. In this way, the spacer sequence serves the remainingpart of the oligonucleotide as a spacer. In a preferred embodiment, anoligonucleotide contains one partial sequence, which has the function ofa primer and is termed a primer sequence, as well as a partial sequence,which is a spacer sequence. As the primer sequences are spaced furtheraway due to the spacer sequences, the nucleic acids to be amplified andthe DNA polymerases can, advantageously, attain a better access to theprimer sequences. In a preferred embodiment, the copies of the originaland of the complement remain attached to the surface of thenanoparticles via the spacer sequences. In a particularly preferredembodiment, the spacer sequences contain restriction sites forrestriction endonucleases such that the synthesised copies can be cutoff the nanoparticles. This preferably takes place after the terminationof the method; however, it can also occur while the method is beingcarried out. That way, the method makes it possible to produce copies ofnucleic acids, which are freely present in the sample. In a preferredembodiment of the method, the spacer sequences are at least as long asthe filling molecules such that the primer sequences are not obscured bythe filling molecules.

In a preferred embodiment, the heat, which is transferred from thenanoparticles to their environment through excitation of thenanoparticles, is sufficient to dehybridise the oligonucleotides on thesurface of the nanoparticles from nucleic acids hybridised to saidoligonucleotides. In this embodiment, the nanoparticles are conjugatedto oligonucleotides and at least a part of the said oligonucleotides ishybridised to at least partially complementary nucleic acids. Throughthe excitation of the nanoparticles, thermal energy is transferred tothe surrounding water, such that, preferably, the temperature of thewater around the nanoparticles is sufficient, dehybridise theoligonucleotides from the nucleic acids bound thereto. In a particularlypreferred embodiment, the method according to the invention is a PCR andthe nanoparticles are conjugated with primers. When carrying out thePCR, preferably double stranded PCR products are formed, in each ofwhich at least one single strand of the double stranded PCR products isconjugated to a nanoparticle. In this embodiment it is advantageouslyachievable to produce the denaturation temperature around thenanoparticles through the excitation of the nanoparticles and to carryout the denaturation of the double-stranded PCR products, withoutheating the entire reaction volume. In this way, the denaturation can beaccelerated, such that the PCR can occur more rapidly. In anotherpreferred embodiment, the annealing temperature and the elongationtemperature are also produced through the excitation of thenanoparticles. In this way, preferably, only a small amount of energyhas to be transferred when compared with the heating of the entire probeto the annealing temperature and elongation temperature. Morepreferably, denaturation, annealing and elongation of the PCR takesplace without a global heating, but exclusively through local heating byexcitation of the nanoparticles. That way, the method can be carried outwithout a device for global heating, such that less equipment isrequired to perform the method.

In another preferred embodiment, the method comprises a global heatingstep. In this, the temperature in at least one step of the method isreached at least partially by global heating. In a more preferablyembodiment of the invention, the method is a PCR and the annealingtemperature is attained by global heating of the reaction volume. Mostpreferably, the reaction volume is heated globally throughout the entiremethod to within a predetermined temperature range, in which annealingtakes place. In this, the elongation temperature and denaturationtemperature are reached through excitation of the nanoparticles.Thereby, advantageously, the device, which produces the global heating,can be implemented in a simple design as it only needs to sustain apredetermined temperature.

In another preferred embodiment, the annealing temperature and theelongation temperature are reached by global heating, exclusively thedenaturation is achieved by the excitation of the nanoparticles. In thisway, advantageously, it can be accomplished that the device that createsthe global heating, needs to only produce a temperature cycle with twodifferent temperatures and can thus be implemented in a simple design.Typically, the elongation and the annealing take place in a narrowtemperature range. As opposed to this, to achieve denaturation, acertain temperature has to be surpassed only. Thus, inhomogeneities inthe excitation of the nanoparticles to produce denaturation are a lesserproblem than in the adjustment of the annealing temperature andelongation temperature. Hence, a preferred embodiment, in which theexcitation of the nanoparticles exclusively serves to producedenaturation, can be technically implemented in a simpler fashion. Thisis particularly true for the preferred case, in which annealingtemperature and elongation temperature are very close to each other,e.g. when the annealing temperature is 60° C. and the elongationtemperature is 72° C., such that the global heating only needs toproduce a small temperature increase.

In an especially preferable embodiment, the annealing temperature is thesame as the elongation temperature. In this case, the method is a PCR.If the annealing temperature is equal to the elongation temperature, atemperature cycle with only two different temperatures is necessary tocarry out the PCR, which means that the method can be performed with asimple setup.

Preferably, the melting temperatures of the primers and the DNApolymerase used are chosen such that at the melting temperature, the DNApolymerase used can still synthesise DNA at a sufficient speed. In anespecially preferred embodiment, the elongation temperature, which isequal to the annealing temperature, is achieved by global heating andthe denaturation is attained through the excitation of thenanoparticles. In this way, the device that produces the global heatingcan be implemented in a simple manner as it only needs to keep onetemperature.

In a preferred embodiment, only a part of the nanoparticles are excitedat any one point during the execution of the method. To this end, e.g.,the means for the excitation of the nanoparticles can be designed insuch a way that they only excite the nanoparticles in one part of thereaction volume. In an especially preferred embodiment, thenanoparticles are excited optically using a laser and the optics, whichguide the light into the reaction volume is designed such that light isonly directed into one part of the reaction volume. The part of thenanoparticles, which is excited, preferably changes during the executionof the method. In other words, a first set of nanoparticles, which isexcited at a first time is not identical with a second set ofnanoparticles excited at a second time. In this case, any number ofnanoparticles can be present in the first and any number ofnanoparticles can be present in the second set as long as the first andthe second set are not identical. Of the two said sets one, e.g., canoverlap with the other, such that the sets form an intersection of thesets. One set can, e.g., be a subset of the other set, such that the oneset contains fewer nanoparticles than the other set. The two sets canalso be modelled in a way that they do not form an intersection, suchthat no nanoparticle is present in the first set as well as the secondset. One of the two sets can also be the empty set such that, e.g., atone time the nanoparticles are excited and at another time, nonanoparticles are excited. In a preferred embodiment, the first and thesecond set essentially contain the same number of nanoparticles.Especially preferably, at different times, a laser excites differentfractions of the nanoparticles. Thereby, in the execution of the method,a laser with a lower power can be employed, which is only justsufficient, to excite a fraction of the nanoparticles. In an especiallypreferred embodiment, two or more lasers are used to excite differentparts of the nanoparticles. This way, advantageously, it is possible toexcite different fractions of the nanoparticles, without requiring anoptical element, which directs the laser to different parts of thereaction volume.

In another preferred embodiment of the invention, a directed movement ofthe sample relative to an exciting field is taking place such that atdifferent times, nanoparticles in different partial volumes of thesample are excited. More preferably, the exciting field is the light ofa laser. In a most preferable embodiment, the light of the laser isdirected by an optical element to excite nanoparticles in differentpartial volumes of the reaction volume at different times. The opticalelement can be moveable, e.g., the optical element can contain a movablemirror, a spatial modulator or an acousto-optical modulator. The laseritself can also be movable. The movement of the sample can beimplemented by moving the reaction vessel, which contains the sample. Inan especially preferred embodiment, the laser beam as well as thereaction vessel is moved. In a further preferred embodiment, the sampleis moved in the reaction volume such that the light of the lasercaptures different partial volumes of the sample at different times.This can, e.g., be achieved by stirring the sample in the reactionvolume, e.g., by using a magnetic stirrer. The reaction volume can,e.g., take an elongated shape, e.g., a channel or a tube. The samplecan, e.g., be moved through a channel with the sample passing a laserbeam in one or several places. Preferably, the sample flows through achannel and passes n positions, at each of which one laser beam isdirected to the sample in the channel; due to the linear flow of thesample across the n laser beams, a PCR with n cycles is performed. Inthis way, the method can be carried out with small amount of movableparts. By using a channel, a miniaturisation, e.g., in the sense of alab on a chip, is possible. Preferably, the laser beam causes thedenaturation while elongation temperature and annealing temperature isproduced by global heating. It is especially preferred that theelongation temperature is equal to the annealing temperature such thatonly one temperature has to be kept through global heating. In this way,the method according to the invention can be performed with minimaleffort.

In a preferred embodiment, a thermolabile DNA polymerase is used in themethod. For the case, in which the excitation of the nanoparticles isused for the denaturation, the exposure of the entire reaction volume tohigh temperatures can be avoided. Rather, it is possible to exclusivelyheat the immediate environment of the nanoparticles to the denaturationtemperature. In this way, the DNA polymerases, which are not located inthe immediate environment, are not exposed to high temperatures.Thereby, it is possible to use DNA polymerases, which are notthermostable, but thermolabile. By the inclusion of thermolabile DNApolymerases, a greater choice of polymerases is available for the methodaccording to the invention. Due to the greater choice of DNApolymerases, the reaction conditions can be varied to a larger extentwhile at the same time maintaining sufficient operation of the DNApolymerase used. In order for the nucleic acids to be amplified to beable to bind to the negatively charged oligonucleotides on thenanoparticles, it may be necessary to use substances, particularlysalts, in the sample at a concentration that can have a detrimentaleffect on the operation of a thermostable DNA polymerase, whichdecreases the efficiency of the method. The greater choice of DNApolymerases—in particular those with a high salt tolerance—can lead toan increase of efficiency of the method. Part of the greater choice ofDNA polymerases are small DNA polymerases such as, e.g., the Klenowfragment and Phi29. In close proximity to the nanoparticles, large,thermostable DNA polymerases may experience steric hindrance due to theattached and possibly already elongated primers. As a result, it may bethat the DNA polymerase does not arrive at the nucleic acid to be copiedor the DNA polymerase is interrupted before it has synthesised acomplete copy of the original or the complement, which causes in adecrease of the efficiency of the method. The greater choice of DNApolymerases, thus, makes an increase in the efficiency of the methodpossible. Due to the greater choice in DNA polymerases, advantageously,enzymes with lower production costs are available as well. The DNApolymerases that are not situated in the immediate environment of thenanoparticles, experience a smaller extent of heat induced deactivation.Thereby, advantageously, a smaller amount of DNA polymerase can be usedin the method.

In a preferred embodiment of the invention, soluble primers as well asprimers on nanoparticles are present in the reaction volume. The solubleprimers are not conjugated to nanoparticles, but are dissolved in thesample. Preferably, the soluble primers are smaller than thenanoparticle primer conjugates and can, thus, exist in a largerconcentration than the nanoparticle primer conjugates. Due to this, thesoluble primers can have a better and faster access to long, doublestranded nucleic acids, such as, e.g., genomic DNA. In an especiallypreferred embodiment, in a first step of the method, the long doublestranded nucleic acids are denatured by global heating of the entirereaction volume, after which the soluble primers hybridise with thenucleic acids. The PCR, at first, runs through one or several cycleswith global heating, during which the DNA polymerase synthesises thedesired, short copies of the long double stranded nucleic acids.Subsequently, the PCR is continued, also using local heating through theexcitation of the nanoparticles.

In a preferred embodiment of the invention, the particle diffusion ofthe nanoparticle primer conjugates can be amplified by using opticalfields. Through optical vortex fields (in accordance with SilviaAlbaladejo et al., Nano Letters, 2009, volume 9, issue 10, pages 3527 to3531, the related content of which forms part of the present disclosureby way of reference), with which the nanoparticles are excited or due tooptical forces (according to Arthur Ashkin et al., Proc. Natl. Acad.Sci., 1997, volume 94, issue 10, pages 4853 to 4860, the related contentof which forms part of the present disclosure by way of reference),which are exerted onto the nanoparticles, the nanoparticle diffusion canbe increased. Thereby, advantageously, a faster hybridisation of thenucleic acid to be amplified with the primers on the nanoparticles cantake place at a given nanoparticle concentration. This can be utilisedto achieve an acceleration of the method according to the invention.

In an embodiment of the invention, the concentration of the products ofan amplification reaction can be detected with test probes. Test probesare nanoparticles, which contain oligonucleotides with test sequences ontheir surfaces. In a preferred embodiment of the method, theoligonucleotides of the test probes have a spacer sequence as a partialsequence. The spacer sequence is situated on the side of theoligonucleotide closer to the nanoparticle. Thus, the spacer sequenceserves the remaining part of the oligonucleotide as a spacer. In apreferred embodiment, the oligonucleotide of the test probes containsboth a partial sequence, which is termed a test sequence, and a partialsequence, which is a spacer sequence. In a preferred embodiment, thetest probes have filling molecules attached thereto. The test sequencescan hybridise with products of the amplification reaction. In this, thetest sequences are preferably at least partially complementary to theproducts of the amplification reaction. In a preferred embodiment, firstnanoparticles are conjugated to forward primers. In the presence of theoriginal and a DNA polymerase, the forward primers are extended suchthat complements are created, which are bound to the first nanoparticlesvia the forward primers. In this, a complement consists of a forwardprimer and an extension sequence, which is created by the extension ofthe forward primer. Especially preferably, a PCR is performed usingsoluble and/or nanoparticle conjugated reverse primers such that in anexponential amplification, preferably a large number of copies of theoriginal and of nanoparticle conjugated complements are produced. Mostpreferably, the first nanoparticles contain on their surface bothforward and reverse primers. In an optional intermediate step, theoriginals and, possibly, their copies are denatured from the complementsthrough local or global heating. The first nanoparticles are thenbrought together with the test probes, if this has not taken placebefore. The test sequences of the test probes are complementary to theextension sequences such that the test probes can bind to the extendedforward primers on the first nanoparticles via the test sequences. Inappropriate reaction conditions, the connection of the firstnanoparticles and the test probes takes place to the extent, in whichthe nanoparticle bound complements are present. This means that if noextension sequences are formed, no connection between test probes andnanoparticles is made. More preferably, the reaction conditions of theamplification and the detection according to the invention using testprobes are chosen such that the extent of the connection of the firstnanoparticles with the test probes allows for a conclusion to be drawnas to what concentration of the original was present in the samplebefore the amplification. Through the connection of the firstnanoparticles to the test probes, a measurable change can arise, e.g., ared shift or broadening of the plasmon resonance in the absorbancespectrum. In an especially preferred embodiment, the measurable change,which occurs through the connection of the test probes with thenanoparticles, is proportional to the concentration of the original inthe sample before the amplification. In this way, advantageously, simpletools can be used to verify the concentration.

In another preferred embodiment, the method comprises forward primers,which are conjugated to first nanoparticles and free and/or nanoparticlebound reverse primers. It is especially preferred for the nanoparticlesto contain forward as well as reverse primers on their surface. In afirst step, a DNA polymerase extends the forward primers to nanoparticlebound complements in the presence of the original. In a second step,starting from the reverse primers, which bind to the nanoparticle boundcomplement, copies of the original are synthesised. After that, thefirst nanoparticles are brought together with the test probes, if thishas not already occurred. In this embodiment, the test sequences arecomplementary to the forward primers. If the forward primers were notextended then the test probes can bind well to the first nanoparticles.If the forward primers were extended then the binding of test sequencesto the forward primers is inhibited due to steric hindrance. If a newlysynthesised copy of the original is hybridised with an extended forwardprimer then the binding of the test sequence to the extended forwardprimer is prevented. In this way, the extent of the connection betweenthe first nanoparticles and the test probes decreases to the extent, inwhich the products of the amplification reaction, i.e., complements andcopies of the original, are synthesised. When choosing the reactionconditions appropriately, the concentration of the original can bedetected such that a measurable change is the smaller the more originalwas present in the sample before the amplification. The measurablechange can, e.g., be a red shift or a broadening of the plasmonresonance in the absorbance spectrum. In this way, advantageously, asimple test can be designed, which allows for the determination ofconcentrations of specific nucleic acids.

The invention makes it possible to provide an improved method for theamplification of nucleic acids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in a)-h) a schematic representation, the nanoparticlesaccording to the invention conjugated to filling molecules, spacersequences and primer sequences.

FIG. 2 shows in a)-e) another schematic representation, thenanoparticles according to the invention conjugated to filling moleculesspacer sequences and primer sequences.

FIG. 3 shows in a schematic representation the setup for performing themethod according to the invention with a laser, a two dimensional mirrorscanner and a sample.

FIG. 4 shows in a schematic representation a further setup for carryingout the method according to the invention with a laser, a mirror, and asample, which is moved relative to the laser beam.

FIG. 5 shows in a schematic representation another setup for carryingout the method according to the invention with a laser, a onedimensional mirror scanner and a sample moved in one dimension.

FIG. 6 shows in a)-f) a schematic representation the nanoparticlesaccording to the invention and the test probes according to theinvention for the positive detection of DNA.

FIG. 7 shows in a schematic representation another setup for carryingout the method according to the invention with a laser, a twodimensional mirror scanner and a sample tube in a water bath.

FIG. 8 shows in two diagrams a) and b) the results of amplificationreactions with global and local heating with test probes for thepositive detection of DNA.

FIG. 9 shows in a)-f) a schematic representation the nanoparticlesaccording to the invention and the test probes according to theinvention for the negative detection of DNA.

FIG. 10 shows in two diagrams a) and b) the results of amplificationreactions with global and local heating with test probes for thenegative detection of DNA.

FIG. 11 shows in a diagram the results of amplification reactions withthe non-thermostable Klenow fragment.

FIG. 12 shows in a diagram the results of amplification reactions with afixed and a moving laser beam.

FIG. 13 shows in a schematic representation a setup for carrying out themethod according to the invention with a light source, a deflectingelement and a movable sample tube.

FIG. 14 shows in a schematic representation a section of a nanoparticleaccording to the invention with filling molecules, oligonucleotides andDNA polymerases.

FIG. 15 shows in four diagrams the results of amplification reactionswith test probes for the negative detection of DNA.

FIG. 16 shows in a schematic representation a first laser for theexcitation of nanoparticles in a sample tube and a second laser and aphoto diode for measuring the transmission of the sample.

DETAILED DESCRIPTION OF THE INVENTION ACCORDING TO SEVERAL EMBODIMENTS

FIG. 1 shows an embodiment of the method according to the invention forthe amplification of nucleic acids 1, which is implemented as PCR. Areaction volume 2 contains first nanoparticles 3. The firstnanoparticles 3 show oligonucleotides 4 on their surface as seen in FIG.1a . One kind of oligonucleotides 4 each contain as a partial sequence aprimer sequence 5 with a sequence A and as an additional, optionalpartial sequence a spacer sequence 6 S. A primer sequence 5 is definedas the sequence of a primer 7. The spacer sequence 6S serves to keep theprimer sequence 5 far enough away from the surface of the nanoparticle 8for a nucleic acid 1 to be amplified to bind to the primer sequence 5with a better efficiency and for the DNA polymerase 10 to find betteraccess to the primer sequence 5. The oligonucleotides 4 with a primersequence 5 A are attached, e.g., via a thiol bond to the surface of thefirst nanoparticle such that the 3′-end is facing away from the firstnanoparticle 3. Optionally, another kind of oligonucleotides 4 can bepresent on the surface of the first nanoparticles 3; these are thefilling molecules 9 F. Using the filling molecules 9, the charge of thenanoparticles 8 can be modulated such that undesired aggregations of thenanoparticles 8 are avoided. Furthermore the filling molecules 9 canincrease the distance of the primer sequences 5 to each other on thesurface of the nanoparticles 8 such that the nucleic acids 1 to beamplified and the DNA polymerase 10 can find a better access to theprimer sequences 5. This can increase the efficiency of the method. Thespacer sequence 6 is preferably at least as long as the filling molecule9 such that, advantageously, the primer sequences 5 protrude from thefilling molecules 9.

A sample 11 is present in the reaction volume 2, which sample 11contains the first nanoparticles 3 from FIG. 1a with the primersequences 5, spacer sequences 6 and filling molecules 9 and, in additionto this, the dNTPs and DNA polymerases 10. A nucleic acid 1 to bedetected can be present in the sample 11. In this embodiment, thenucleic acid 1 to be detected is a DNA single strand, which is alsotermed the original 12 and contains a partial sequence A′ as well as apartial sequence B′. The original 12 can contain further partialsequences, e.g. as overhang on the 5′ or 3′-end or between the twopartial sequences A′ and B′. In FIG. 1b , the original 12 binds with itspartial sequence A′ to the primer sequence 5 A on the surface of thefirst nanoparticle 3. In FIG. 1c , it is shown that a DNA polymerase 10binds to the original 12 and to the primer sequence 5 A hybridised tothe original 12. Subsequently, the DNA polymerase 10 synthesises in anelongation step, which is shown in FIG. 1d , starting from the 3′ end ofthe primer sequence 5 A a nucleic acid 1 complementary to the original12, which nucleic acid is termed the complement 13 and is connected to aspacer sequence 6 on the surface of the first nanoparticle 3. In FIG. 1e, the first nanoparticle 3 is then irradiated with light, which isabsorbed by the first nanoparticle 3 on account of its plasmonic ormaterial properties and which is transformed into heat. The heat istransferred to the environment of the first nanoparticle 3 and withinthe region of the original 12 and the newly synthesised complement 13hybridised to the original 12 is sufficient for the original 12 todenature from the complement 13. The original 12 is now free again, asshown in figure if such that it can bind to another primer sequence 5and further nanoparticle bound complements 13 can be synthesised inadditional cycles of the method. In this way, a linear increase of theconcentration of the complements 13 is created with an increasing numberof cycles. The steps of the method described in FIGS. 1g and 1h areclarified in this document further below.

FIG. 2 shows an embodiment of the method according to the invention, inwhich the nanoparticles 8 are situated in a sample 11. The nanoparticles8 show filling molecules 9 F on their surface. Furthermore, thenanoparticles 8 are conjugated to oligonucleotides 4. A first kind ofoligonucleotides 4 consists of a spacer sequence 6 S and a primersequence 5 A. A second kind of oligonucleotides 4 consists of a spacersequence 6 S and a primer sequence 5B′. In this embodiment, the original12 to be amplified is a single stranded DNA molecule with the partialsequences A, C, B (not shown). Starting from a primer sequence B′ on thesurface of the nanoparticle 8, a DNA polymerase 10 synthesises a strandcomplementary to the original 12 such that, as shown in FIG. 2a , a DNAsingle strand with the sequence S, B′, C′, and A′ is situated on thenanoparticle 8. At the same time, it can be seen on FIG. 2a that a DNApolymerase has synthesised a copy of the original 12 starting from theprimer sequence 5 A, which is connected with the spacer sequence 6 S onthe surface of the nanoparticle 8. As shown by the arrow in FIG. 2a ,the copy of the original 12 attached to the nanoparticle 8 hybridiseswith its partial sequence B to a primer sequence 5 B′ on the surface ofthe same nanoparticle 8. A second arrow in FIG. 2a shows that thecomplement 13 synthesised on the surface of the nanoparticle 8hybridises with its partial sequence A′ to a primer sequence 5 A on thesurface of the same nanoparticle 8. The result of the two saidhybridisations is shown in FIG. 2b . In this, the original 12 as well asthe complement 13 form a loop on the surface of the nanoparticle 8. FIG.2c shows that a strand complementary to the original 12 is synthesisedstarting from the primer 7 B′, which strand is connected to the surfaceof the nanoparticle 8 via a spacer sequence 6 S. Another DNA polymerase10 synthesises a copy of the original 12 starting from the primersequence 5A, which copy is also connected to the surface of thenanoparticle 8 via a spacer sequence 6. The result of the two synthesesis shown in FIG. 2d . In this embodiment, the forward primer 14 as wellas the reverse primer 15 are situated on the same nanoparticle 8. Inthis way, a newly synthesised DNA strand can hybridise back to a primer7 on the same nanoparticle 8. This can lead to the acceleration of themethod according to the invention as the newly synthesised DNA stranddoes not have to travel far to meet a complementary primer 7. Rather,the newly synthesised DNA strand can bind particularly rapidly to acomplementary primer 7 on the surface of the same nanoparticle 8, whichis facilitated especially by the high local concentration of the primer7 on the nanoparticle 8. After the excitation of the nanoparticle 8 inFIG. 2d , e.g., with a laser 16, the copies of the original 12 and thecopies of the complement, which are each attached to the surface of thenanoparticle 8 via spacer sequences 6, dehybridise. After that, a copyof the original 12, which is attached to the nanoparticle 8, canhybridise with a complement 13, which is attached to the surface ofanother, identical nanoparticle 8. Through the hybridisation, thenanoparticles 8 are connected, such that a measurable change occurs. Themeasurable change can, e.g., consist in a colour change of the sample11. The embodiment of the method according to the invention shown inFIGS. 2a to 2e makes it possible to provide a simple test, which servesto detect the original 12.

FIG. 3 shows a setup, which is suitable for carrying out the methodaccording to the invention. The setup contains a light source 17, whichin this case is implemented as a laser 16, and a two dimensional mirrorscanner 18, which can direct light from the laser 16 to the sample 11.The two dimensional mirror scanner 18 can deflect the laser in twodimensions. In this setup, the denaturation in the sample 11 occurs byfocussing a laser beam onto a part of the sample 11. During the method,the laser beam is deflected such that it hits different parts of thesample 11. In the example shown in FIG. 3, the laser beam is deflectedby the mirror scanner 18 such that the laser beam moves through thereaction volume 2, in which the sample 11 is situated, row by row. InFIG. 3, the path followed by the laser beam in the sample 11 is shown ina dashed line. Due to the fact that at any one time during the method,only parts of the sample 11 are excited, lasers 16 with a smaller poweroutput can be used. As excitations of under one microseconds aresufficient to denature DNA with the aid of optothermally heatednanoparticles 8, a typical focus diameter of a laser 16 of approximately10 to 100 μm allows a laser beam to scan the sample 11 at a speed ofapproximately 10 to 100 m/s while leading to a denaturation of the DNAat each point that the laser sweeps across. This enables a very fastscanning of large sample volumes. The complete scanning of an area of 1cm² takes, e.g., only 128 ms at a focus diameter of 78 μm and 128 rowsat an inter-row distance of 78 μm and a row length of one centimetre ata velocity of the scanning laser beam of 10 m/s. Advantageously, this issignificantly shorter than a denaturation step using global heatingwould generally require. Optical elements such as, e.g., a mirrorscanner 18 shown in FIG. 3 and so called F-theta-lenses can achieve agood homogeneity of the focus quality and size across the entire sample11 scanned. As an alternative to a continuously emitting laser 16, apulse laser 16 or a thermic radiator can be used.

FIG. 4 shows a setup for carrying out the method according to theinvention in which a laser 16 and a mirror 19 are fixed and the laserbeam of the laser 16 is directed towards the sample 11 using the mirror19. In this, the sample 11 is arranged to be movable in two dimensionssuch that by moving the sample 11 the entire sample 11 or large parts ofthe sample 11 can be reached by the focus of the laser 16.

FIG. 5 shows a setup for carrying out the method according to theinvention, in which a laser 16 is fixed, and a mirror scanner 18 candeflect the laser beam of the laser 16 in one direction. The sample 11is arranged to be movable in one direction such that by moving themirror scanner 18 and the sample 11 the entire sample 11 or large partsof the sample 11 can be reached by the laser beam. One possibility fordetecting a nucleic acid 1 by PCR according to the invention is shown inFIG. 6. In this, first nanoparticles 3, which show filling molecules 9 Fand first oligonucleotides 20 on their surface, are situated in asample. The first oligonucleotides 20 consist of a spacer sequence 6 Sand a primer sequence 5 A, as shown in FIG. 6a . If an original 12 withthe partial sequences A′ and B′ is present in the sample 11 then theoriginal 12 hybridises to the complementary primer sequence 5 A on oneof the first nanoparticles 3. A DNA polymerase 10 synthesises thecomplement 13 with the partial sequences A and B, starting from theprimer sequence 5 A such that the complement 13 is connected to thesurface of the first nanoparticle 3 via the spacer sequence 6 S, asshown in FIG. 6c . In a next step, the test probes 21 shown in FIG. 6dare added to the sample. The test probes 21 are second nanoparticles 22,which shown filling molecules 9 and second oligonucleotides 23 on theirsurface. The second oligonucleotides 23 contain a spacer sequence 6 Sand a test sequence 5 B′. The test sequence 5 B′ can hybridise with thecomplementary partial sequence B of the complement 13 on the surface ofthe first nanoparticle 3, as shown in FIG. 6f . Thereby, the firstnanoparticles 3 and the second nanoparticles 22 are connected such thata measurable change can occur. If the original 12 is not present in thesample 11, then no complement 13 is created on the surface of the firstnanoparticle 3, as seen in FIG. 6b . As there is no complement 13 on thefirst nanoparticles 3, first nanoparticles 3 and second nanoparticles 22cannot connect to each other and the measurable change does not occur.In this embodiment the sequence B′ is complementary to the sequence B;the sequence B′ can also be complementary to parts of the sequence A.The spacer sequence 6 S on the first nanoparticles 3 is identical to thespacer sequence 6 S on the second nanoparticles 22. In a furtherembodiment however, different spacer sequences 6 can be used on thefirst nanoparticles 3 and the second nanoparticles 22. Also, severaldifferent spacer sequences 6 can be used on the same kind ofnanoparticles 8. The buffer and hybridisation conditions, e.g.,temperature, salt concentrations, nanoparticle concentrations,concentrations of additional buffer additives, pH, are preferably chosensuch that a hybridisation connecting the first nanoparticles 3 with thesecond nanoparticles 23 can only arise after the completed extension ofthe primer sequence 5 A on the first nanoparticles 3. The connection ofthe first nanoparticles 3 with the second nanoparticles 22 can, e.g., bedetected as a red shift and broadening of the plasmon resonance in theabsorbance spectrum. The connection can also be detected, e.g., bymeasuring the change in transmission at one or several wavelengths afteroptothermal excitation of the nanoparticles 8 and the resultantdenaturation of the nucleic acids 1, which connect the firstnanoparticles 3 with the second nanoparticles 22. The test probes 21 canbe supplied in a special hybridisation buffer to which at least a partof the sample 11, which contains the first nanoparticles 3, is addedafter the step of the method, in which the synthesis of the complement13 is enabled. The test probes 21 can, together with the firstnanoparticles 3, be present in the sample already before the start ofthe method. In this case, the test probes 21 can be passivated such thatthey do not act as primer 7. The passivation of the test probes 21 canconsist in choosing the primer sequence 5 on the test probes 21 in suchway, that no hybridisation of the said primer sequence 5 with theoriginal 12 occurs at the annealing temperature during the PCR, but onlyafter subsequent lowering of the temperature. The passivation of thetest probes 21 can be created by attaching the second oligonucleotide23, which contain partial sequences of the original 12, at the 3′-end ofthe second nanoparticles 22 such that the DNA polymerase 10 cannotextend the second oligonucleotide 23. In this case, the secondoligonucleotides 23 can be free on their 5′-end or connected to thesecond nanoparticles 22. The test probes 21 can also be passivated by abase modification, e.g., with dideoxy cytosine (ddC) at the free primeend of the second oligonucleotide 23, which prevents elongation.

In the embodiment of the method as shown in FIG. 6, first nanoparticles3 made of gold and with a diameter of 60 nm are functionalised witholigonucleotides 4 (according to J. Hurst et al., Anal. Chem., 78(24),8313-8318, 2006, the related content of which forms part of the presentdisclosure by way of reference). In this, one part oligonucleotide 4 ID1and—as a filling molecule 9—four parts oligonucleotide 4 ID 2 are used.After functionalisation and six washing steps, the first nanoparticles 3are present in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20,0.01% azide, 1 mM EDTA, pH 7.5) at a concentration of 200 μl. Theamplification reaction is performed in a total volume of 10 μl in 200 μlsample tubes 24 (5 μl DreamTaq PCR Mastermix 2× (fermentas), 0.1 μl NaCl5 M, 0.1 μl MgCl₂ 250 mM, 0.1 μl MgSO₄ 250 mM, 1 μl of thefunctionalised first particles 200 pM, 1 μl oligonucleotide 4 ID3 (as anoriginal 12 to be amplified, wherein the concentration of the original12 to be determined is in a total volume of 10 μl, e.g., 0 pM, 10 pM, 20pM or 50 pM) dissolved in water with 100 nM oligonucleotide 4 ID4(oligonucleotide 4 ID4 serves to saturate surfaces e.g., during thestorage of the original 12 before the reaction), 2.7 μl water). As shownin FIG. 7, the sample tubes 24 are brought to a temperature of 65° C. ina glass cuvette 25 in a water bath 26, wherein said temperature is theannealing as well as the elongation temperature. The water bath 26serves—in addition to keeping the correct temperature—also the bettercoupling of the laser 16 into the non-planar surface of the sample tubes24. The water in the water bath 26 enables the reduction of thedifference in the refractive indices between the outside of the sampletubes 24 and its inside, which is filled with the PCR reaction mixture;thus a refraction of the laser beam and a resultant negative influenceon focus quality and sharpness is suppressed. Thereby, advantageously,the coupling of the laser 16 is improved. The laser 16, which serves toexcite the nanoparticles, is a frequency-doubled diode-pumped: Nd:YAglaser (Coherent Verdi V10) which is focussed into the sample tubes 24 inthe water bath 26 (focal diameter approximately 20 μm) with an outputpower of 1.5 W with a F-theta-lens (Jenoptik, focal distance 100 mm)behind a mirror scanner 18 (Cambridge technologies, Pro Series 1). Themirror scanner 18 allows to move the focus row by row through the sampletubes 24, as already shown in FIG. 3, and thus to involve the entire PCRreaction volume in the optothermal amplification. Per sample tube 24,400 rows are scanned with the focus at a distance of approximately 12 μmat a row speed in the sample tubes 24 of approximately 2 m/s. Thiscorresponds to one cycle in the first sample tube 24. Subsequently, allthe other sample tubes 24 are scanned one after the other, such thateach sample tube 24 has experienced one cycle. After a waiting time of40 s after the scanning of the first sample tube 24, the next cycle isstarted and this is repeated until each sample tube 24 has completed 25cycles. As a starting concentration of the original 12 in the firstsample tube 24, 0 pM, in the second sample tube 24 20 pM and in thethird sample tube 24 50 pM is chosen. For the negative control, a fourthsample tube 24 is inserted into the water bath 26, which also containsthe original 12 at a concentration of 50 pM, but is not hit by the laserbeam. After the first, the second and third sample tubes 24 havecompleted 25 cycles, all four sample tubes 24 are removed from the waterbath 26. To examine the effect of the laser cycles and the concentrationof the original 12, a test probe 21 is used, which is able toexclusively hybridise to the test sequences produced through theextension of the nanoparticle bound primers under the chosen buffer andhybridisation conditions. In this, the extension of the primer 7 iscomplementary to the original 12, as shown in FIG. 6c . To produce thetest probes 21, second nanoparticles 22 made of gold and with a diameterof 16 nm are functionalised with oligonucleotides 4 (according to J.Hurst, supra). Therein, one part oligonucleotide 4 ID5 and—as a fillingmolecule 9—four parts oligonucleotides 4 ID2 are used. After thefunctionalisation and six washing steps, the second nanoparticles 22 arepresent in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01%azide, 1 mM EDTA, pH 7.5) at a concentration of 200 pM. For thehybridisation of the oligonucleotides 4 on the first nanoparticles 3with the oligonucleotides 4 on the second nanoparticles 22, a modifiedphosphate buffer is used (13 mM PBS, 200 mM NaCl, 0.02% Tween 20, 1 mMEDTA, 20 mM sodium citrate, 1 μg/ml PVP10, pH 7.5). 10 μl hybridisationsolution contain 2.25 μl of the modified phosphate buffer, 3 μlformamide, 2 μl NaCl 5M, 0.25 μl of the 200 pM test probe solution and2.5 μl of the corresponding PCR solution from the optothermalamplification, which contains the first nanoparticles 3. If a sufficientamount of the original 12 with the sequence ID3 was present in thesample tube, the oligonucleotide 4 with the sequence ID1 on the surfaceof the first nanoparticle 3 is extended and is able to hybridise withthe oligonucleotide 4 with the sequence ID5 on the surface of the testprobe, as shown in FIG. 6f . The hybridisation is verified usingoptothermal excitation of the nanoparticles 8 (according to EP 2162549,the related content of which forms part of the present disclosure by wayof reference). To this end, the sample tubes 24, as shown in FIG. 16,are hit with pulses from a first laser 27 (50 μs pulse duration, 532 nmwavelength, approximately 700 mW peak power, focus diameterapproximately 30 μm). Thereby, the nanoparticles 8 are optothermallyheated and transfer heat to their environment. If first nanoparticles 3and second nanoparticles 22 are connected due to the hybridisation ofoligonucleotides 4, as is shown in FIG. 6f , then they will be separatedby the laser pulse. This can be detected using a second laser 28(wavelength 630 nm, power 5 mW continuously) as shown in FIG. 16; thefocus of the second laser (30 μM diameter) is superimposed with thefocus of the first laser 27, which is preferably used fordehybridisation exclusively, the focus of the second laser detects theabsorbance before and after the laser pulse of the first laser 27. Theoptical path on which the change in absorbance is induced optothermallyand is measured amounts to approximately 2 mm. The intensity of thelight of the second laser 28 transmitted through this layer is measuredwith a photo diode 35. The optothermally induced transmission change isdetermined from the difference of the current in the photo diode beforeand after the pulse, which transmission change is produced by thedehybridisation of the extended first oligonucleotides 20 and secondoligonucleotides 23 between the nanoparticles 8 and the subsequentdiffusion of the nanoparticles away from each other.

FIG. 8a shows the relative transmission change, which is produced by thelaser pulse of the first laser 27 and the resultant dehybridisation ofthe oligonucleotides 4 between the first nanoparticles 3 and secondnanoparticles 22; the relative transmission change is a measure for thepresence of gold-DNA-gold-bonds in the sample tubes 24. Below thediagram in FIG. 8a , the number of completed cycles is shown in a firstrow. In a second row, which is situated underneath the first row, theconcentration of the original 12 in the sample tube 24 before carryingout the amplification is shown in pM. On the right side of the diagramin FIG. 8a in the section B, the first, second and third sample tube 24are shown from left to right, each of which has completed 25 optothermalcycles; in addition to this, the fourth sample tube 24, which has notreceived any optothermal treatment, is shown. It can clearly be seenthat the measured transmission change as an indicator for the gold-DNAgold-bonds increases with the increasing concentration of the original12 before the amplification when the 25 cycles have been completed. Forthe first sample tube 24 without original 12 and the fourth sample tube24 without optothermal treatment, only a small transmission change isobserved. This shows that, herein, no extension of the primer sequences5 on the first nanoparticles 3 has taken place and thus, no binding tothe test probe is possible. Only after completing the optothermal cyclesand in the presence of the original, an extension of the primersequences 5 on the first nanoparticles 3 can be created by the DNApolymerase 10, which leads to a connection of the first nanoparticles 3with the second nanoparticles 22 and finally to a transmission change asa result of the optothermally induced separation of the nanoparticles 8.

As a comparison, FIG. 8a shows in section A on the left side the resultof a corresponding experiment, which did not heat the DNA locallythrough optothermal excitation of the nanoparticles 8, but heated theentire reaction volume 2 globally in a conventional thermal cycler(Labnet Multi Gene II). From left to right, the first to fourth sampletubes 24 are shown, the content of which is identical to the one in theexperiment described in the previous paragraph. First, second and thirdsample tubes 24 were subjected to a classical PCR protocol (93° C. for 1s, 53° C. for 20 s, 35 cycles). As in the case of the optothermalheating, it can be observed that the more of the original 12 is presentin each sample tube 24 before the amplification, the larger is thetransmission change measured, which is created by the laser pulse andthe resultant dehybridisation of DNA between the first nanoparticle 3and the second nanoparticle 22 and which transmission change is themeasure for the presence of gold-DNA-gold bonds in the solution. Thefourth sample tube 24, while containing 50 pM of the original 12, hasnot been heated cyclically and shows almost no transmission change. Inthis case, the primer sequences 5 on the first nanoparticles 3 were notextended to a sufficient degree.

FIG. 8b shows a similar experiment with global heating of the entirereaction volume 2, however, the concentration of the original 12 in thesample tubes 24 is constant at 10 pM before the amplification (secondrow below the diagram), while the number of the cycles is increasing(first row below the diagram). Here, it can clearly be seen that with anincrease in the number of cycles, the transmission change measuredbecomes larger, which is a clear sign that the more primer 7 on thefirst nanoparticles 3 are extended, the more cycles are completed andthus a clear sign that the origin of the signal measured is indeed thecompleted elongation of the oligonucleotides 4 on the firstnanoparticles 3 by the DNA polymerase 10.

In an embodiment of the method, a free reverse primer 15, which binds tothe 3′-end of the complement, is used after the elongation of the primersequence 5 on the surface 4 of the first nanoparticles 3, during whichextension a nanoparticle bound complement 13 is produced. FIG. 1g shows,that the complement 13 with the partial sequences A and B alreadysynthesised, which is connected to the surface of the first nanoparticle3 via a spacer sequence 6, hybridises with a primer 7 B′, which waspreviously freely present in the sample 11. In this, the primer 7 hasthe sequence B′ and is connected with the partial sequence B of thecomplement 13. Starting from the primer 7 with the sequence B′, the DNApolymerase synthesises a copy of the original 12. In FIG. 1g it is alsoshown that the original 12 has bound to another primer sequence 5 A onthe surface of the first nanoparticle 3 and a DNA polymerase 10synthesises another complement starting from the primer sequence 5 A.The original 12, the copy of the original 12 and the two complements 13connected with the first nanoparticle are shown in FIG. 1h . Asubsequent denaturation through excitation of the first nanoparticles 3results in the original 12 and its copy becoming free. In this, theoriginal 12 as well as its copy can serve as a template for theamplification in subsequent steps of the method. After a waiting time,which might be necessary for the hybridisation of the original 12 andcopies of the original 12 with the primer sequences 5 A on the firstnanoparticles 3 and of free primers B′ with the primer sequences 5already elongated on the first nanoparticles 3, the next cycle of themethod can be performed with another excitation of the firstnanoparticles 3. Preferably, this cycle is repeated until a sufficientamount of extended primer sequences 5 are present on the firstnanoparticles 3 and/or a sufficient amount of copies of the original 12are present in the sample 11 to allow a verification of theamplification effected or, respectively, the presence of the original 12in the sample 11. By using a free primer 7 B′, as shown in FIGS. 1g and1h , an exponential amplification of the original 12 is possible. InFIG. 1a to 1f , only a linear amplification of the nanoparticle boundcomplement 13 is achievable without this free primer 7. The denaturationof DNA can, in one embodiment, take place in less than one millisecond.Even at 40 cycles, the denaturation of the DNA and the subsequentcooling to the elongation temperature only require a few milliseconds intotal in this embodiment. This means that the duration of the methodaccording to the invention is not determined by technical limitationssuch as the heating and cooling rate of conventional thermocyclers.Also, the thermalisation times in the reaction volume 2 are avoided asthe heat is always produced in the environment of the nanoparticles 8and an equilibrium temperature distribution is effected withinnanoseconds. Thereby, a PCR can be accelerated significantly.

One possibility to verify the achieved amplification is shown in FIG. 9.FIGS. 9a and 9c outline the exponential amplification using a dissolvedreverse primer 7 B′ as already shown in FIG. 1a to 1h . After that, thetest probes 21 are added to the sample 11. In this embodiment, the testprobes 21 consist of second nanoparticles 22, which are functionalisedon their surface with optional filling molecules 9 and the test sequenceA′, as shown in FIG. 9d . Optionally, a spacer sequence 6 S, which isnot necessarily identical to the spacer sequence 6 S on the firstnanoparticles 3 from FIG. 1 or FIG. 9a , can be placed between the testsequence A′ and the surface of the second nanoparticles 22. The testsequence A′ is complementary to at least a part of the primer sequence 5A on the first nanoparticles 3. The test sequence A′ competes for theprimer sequence 5 A with the copies of the original 12 containing thepartial sequence A′ produced in the method in FIG. 1a to 1h . This meansif many copies of the original 12 are present then the primer sequences5 A on the surface of the first nanoparticles 3 are already occupiedwith the partial sequences A′ of the copies of the original 12. In thiscase, the primer sequences 5 A cannot hybridise or can only hybridise toa limited extent to the test probes A′ on the second nanoparticles 22.Thus, the first nanoparticles 3 are not connected or only connected to alimited extent to the second nanoparticles 22. As shown in FIG. 9c , theelongated primer sequences 5 A on the first nanoparticles 3 arehybridised with the original 12 and its copies and thus form rigid,double-stranded DNA which can pose a steric hindrance; due to this,also, a connection of the first nanoparticles 3 to the secondnanoparticles 22 is prevented when a high number of copies of theoriginal 12 are present. In the absence or presence of a small number ofthe original 12 and copies of the original 12, the first nanoparticles 3are predominantly present with unoccupied primer sequences 5 A, as isshown in FIG. 9b . When the test probes 21 are added, the secondnucleotide 23 A′ hybridises to the unoccupied primer sequences 5 A onthe first nanoparticles 3. Due to this, the first nanoparticles areconnected to the second nanoparticles 22, as shown in FIG. 9e . In thisembodiment, the extent of the connection of the first nanoparticles 3 tothe second nanoparticles 22 is the weaker, the more copies of theoriginal 12 have been produced by the amplification reaction, whichdepends on the concentration of the original 12 at the start of theamplification reaction. The buffer- and hybridisation conditions (e.g.temperature, salt concentration, nanoparticle concentration,concentrations of further buffer additives, pH) are chosen such thatafter completed specific extension of the primer sequence 5 A andcompleted synthesis of copies of the original 12 the suppression of thehybridisation of the primer sequences 5 A with the secondoligonucleotide 23 A′ is as efficient as possible. At the same time, thesaid conditions are chosen such that when no amplification has takenplace, an efficient hybridisation of the primer sequences 5 A with thesecond oligonucleotides 23 A′ is created. The connection of the firstnanoparticles 3 with the second nanoparticles 22 resulting from thehybridisation can be verified by, e.g., a red shift and broadening ofthe plasmon resonance in the absorbance spectrum or by measuring thetransmission change at one or several wavelengths after optothermalexcitation of the nanoparticles 8 and the resulting denaturation of thenanoparticle linking DNA. Alternatively, the verification or aquantification of the copies of the original 12 produced in the methodcan be performed, e.g., by PCR, real-time PCR, quantitativereal-time-PCR, gel-electrophoresis or by using dye labelled probes.

In the embodiment of the method shown on FIG. 9, first nanoparticles 3made of gold and with a diameter of 60 nm are functionalised witholigonucleotides 4 as already shown in the embodiment in FIG. 6, theratio of oligonucleotide 4 ID 1 to oligonucleotide 4 ID2 in FIG. 9,however, is 1:9. After functionalisation and six washing steps, thefirst nanoparticles 3 are present in a concentration of 200 pM in a PBSbuffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01% azide, 1 mM EDTA,pH 7.5). The amplification reaction is carried out in a total volume of10 μl in 200 μl sample tubes 24 (5 μl DreamTaq PCR Mastermix 2×(fermentas), 0.1 μl NaCl 5 M, 0.1 μl MgCl₂ 250 mM, 0.1 μl MgSO₄ 250 mM,1 μl of the functionalised first nanoparticle 3 200 pM, 1 μl reversedprimer IDG 500 nM, 1 μl oligonucleotide 4 ID3 (as original 12 to beamplified, wherein the concentration of the original 12, which is to bedetermined in the total volume of 10 amounts to, e.g., 0 pM or 10 pM)solved in water with 100 nM oligonucleotide 4 ID4 (herein,oligonucleotide 4 ID4 serves the saturation of surfaces, e.g., duringstorage of the original 12 before the reaction), 1.7 μl water). Thesample tubes 24 are kept at a temperature of 54° C. in a glass cuvette25 in a water bath 26, as shown in FIG. 7.

In this, 54° C. constitutes the annealing temperature as well as theelongation temperature. The water bath 26 serves, in addition to thetemperature control, also to better couple the laser 16 into thenon-planar surface of the sample tubes 24. The water in the water bath26 permits for the difference in the refractive index between theoutside and the inside of the sample tube 24 filled with the PCRreaction mixture to be reduced and thus to supress a refraction of thelaser beam and a resultant negative influence on the focus quality andsharpness. Thereby, advantageously, the coupling of the laser 16 isimproved. The laser 16, which serves to excite the nanoparticles 8, isfrequency doubled diode-pumped Nd:YAg-Laser (Coherent Verdi V10), whichis focussed at an output power of 3 W with an F-theta-lens (Jenoptik,focal distance 100 mm) behind a mirror scanner 18 into the sample tubes24 in the water bath 26. The mirror scanner 18 permits for the focus tobe moved row by row through the sample tubes 24, as shown in FIG. 3 andthus to involve the entire reaction volume 2 in the optothermalamplification. Per sample tube 24, 400 rows with a distance aufapproximately 12 μm are scanned with the focus at a row-velocity in thesample tube 24 of approximately 2 m/s. This corresponds to one cycle inthe first sample tubes 24. Subsequently, all other sample tubes 24 arescanned one after the other such that each sample tube 24 hasexperienced one cycle. After a waiting period of 40 s after the scanningof the first sample tube 24, the next cycle is started, this is repeatedaccording to the predetermined number of cycles. 7 sample tubes 24 areexamined, which are shown in FIG. 10a from left to the right. The firstand second sample tube 24 do not contain any original 12. In the thirdtill seventh sample tube 24, 10 pM of the original 12 are present as aninitial concentration. As a control, the first and third sample tubes 24are not treated optothermally. The fourth sample tube 24 was treatedwith 5 cycles, the fifth sample tube 24 with 15 cycles and the sixthsample tube 24 was treated with 25 cycles optothermally. The second andseventh sample tube 24 were treated with the maximum number of 35 cyclesoptothermally. All sample tubes 24 are inside the water bath 26 for thesame amount of time, only the optothermal excitation differs. After thesecond and seventh sample tube 24 have completed all 35 cycles, allseven sample tubes 24 are removed from the water bath 26. One advantageof the method is that without great effort, different samples 11 can betreated with a different number of cycles, this can, e.g., be applied ina parallelised quantitative PCR.

The test probe 21 serves to determine the effect of the laser cycles andthe concentration of the original 12, which test probe 21 can—under thechosen primer- and hybridisation conditions—preferably hybridise to thefirst nanoparticles 3 functionalised with primer sequences 5, whichnanoparticles are not blocked by complementary copies of the original12, which copies were produced in the amplification reaction. Thiscorresponds to the test probe 21 as shown in FIG. 9. To produce the testprobe 21, second nanoparticles 22 made of gold and with a diameter of 60nm were functionalised with oligonucleotides 4 (according to J. Hurst,supra). In this, four parts oligonucleotide 4 ID2 and one partoligonucleotide 4 ID7 are used. After functionalisation and six washingsteps, the second nanoparticles 22 were present at a concentration of200 pM in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01%azide, 1 mM EDTA, pH 7,5). For the hybridisation, a modified phosphatebuffer was used (13 mM PBS, 200 mM NaCl, 0.02% Tween 20, 1 mM EDTA, 20mM sodium citrate, 1 μg/ml PVP10, pH 7.5). 10 μl hybridisation solutioncontain 5.75 μl of the modified phosphate buffer, 1.5 μl formamide, 0.25μl of the 200 pM test probe solution and 2.5 μl of the correspondingPCR-solution of the optothermal amplification reaction, which containsthe first nanoparticles 3. The detection of the connection between thefirst nanoparticles 3 and the second nanoparticles 22 occurs byoptothermal excitation of the nanoparticles 8, as described in FIG. 8a .FIG. 10a shows the change in transmission, which is produced by thelaser pulse and the resultant dehybridisation of DNA between the firstnanoparticles and second nanoparticles 22 and which transmission changeis a measure for the presence of gold-DNA-gold-bonds in the sample 11.Shown in FIG. 10a on the left side in the section A are the first andthe second sample tubes 24, which each contain no original, wherein thefirst simple tube 24 has completed none and the second simple tube 24has completed 35 optothermal cycles. Both sample tubes 24 show a highmeasured transmission change as an indicator for a high measure ofgold-DNA-gold-bonds. Without original 12, the different number ofcompleted cycles thus has, in this case, no influence on thetransmission change measured or on the measure of gold-DNA-gold-bonds;this is because no original 12 was available for the amplification andthus no blockage of the primer sequences 5 by copies of the original 12can take place.

FIG. 10a shows on the right side in the section B the third till seventhsample tube 24 with an increasing number of optothermal cycles in theamplification reaction at an initial concentration of the original 12 of10 pM. Here, it is evident that the transmission change measured as anindicator for the gold-DNA-gold-bonds essentially decreases with anincreasing number of completed optothermal cycles. This shows that themore copies of the original 12 are produced, the more cycles arecompleted. In this, it is noteworthy that at an initial concentration ofthe original 12 of 10 pM, twice as many first nanoparticles 3 asoriginals 12 are present in the sample 11. Each first nanoparticle 3typically carries between 1000 and 10000 primer sequences 5. In theinitial concentration of the original 12, thus, approximately 1 in 2000primer sequences 5 are blocked, which does not lead to a significantsuppression of the gold-DNA-gold bonds between the first nanoparticles 3and second nanoparticles 22. An effective suppression of thegold-DNA-gold-bonds, as shown in FIG. 10a with an increasing optothermalnumber of cycles, is only possible through a considerable amplificationof the low initial concentration of the original 12.

FIG. 10b shows in an alternative detection method the concentration ofthe copies of the original 12 after the amplification reaction. In this,the samples 11 from the seven sample tubes 24 from FIG. 10a are firstdiluted 50-fold in water and subsequently a real-time PCR is carriedout, which allows for the quantitative detection of the copies of theoriginal. To this end, a real-time PCR solution is used, wherein 10 μlcontain 5 μl 2× Phusion Blood PCR-buffer (including dNTPs and MgCl₂;Biozym), 0.2 μl Phusion Blood polymerase, 1 μl SybrGreen I (10×; Roche),1 μl primer ID5 5 μM and 1 μl primer ID8 5 μM, 0.8 μl H₂O and 1 μl ofthe 50-fold diluted sample 11. For the real-time PCR, at first adenaturation at 98° C. is carried out for 1 minute, subsequently, 40cycles are completed, which each consist of 1 second at 98° C., 5seconds at 66° C. and 1 second at 72° C. The fluorescence of theSybrGreen I is measured at the end of each annealing phase at 66° C. Forthe real-time PCR, a Stratagene Mx3005P by Agilent Technologies wasused. On the y-axis in FIG. 10b , the threshold cycle (Ct-value) of thereal-time PCR is shown, in which the copies of the original have reacheda predetermined concentration for the first time. The higher theconcentration of the copies of the original 12 at the start of thereal-time PCR, the smaller is the threshold cycle. The results in FIG.10b confirm the results from 10 a: the more optothermal cycles arecompleted at a given initial concentration of the original 12, thehigher is the number of copies of the original 12 after theamplification reaction.

Preferably, in the optothermal denaturation, only small partial volumesof the sample 11 are heated such that it is also possible to usenon-thermostable DNA polymerases 10. In one embodiment, the Klenowfragment 29, which is not thermal-stable, is used as DNA polymerase. TheKlenow fragment 29 has the advantage that it is more salt tolerant inthe amplification reaction. Thereby, preferably better reactionconditions can be chosen in detection reactions with first nanoparticles3 and second nanoparticles 22, which can lead to better specificity andsensitivity of the detection reaction. In addition to this, the Klenowfragment 29 offers the advantage that at 76 kDA, it is smaller than thetypically used Taq DNA polymerase with 95 kDA. Thus, in the closedproximity of nanoparticles 8 functionalised with oligonucleotides 4, theKlenow fragment 29 experiences less steric hindrance than the Taq DNApolymerase 10. Furthermore, the Klenow fragment 29 offers the advantagethat its optimal elongation temperature is at 37° C. The elongation at37° C. offers the advantage that a smaller thermal strain is exerted onthe nanoparticles 8 and thus lower requirements for the stability of thenanoparticles 8 are necessary; at the same time there, is moreflexibility in the use of potentially nanoparticle destabilizing salts.In this embodiment, in which the Klenow fragment 29 is used foramplification, first nanoparticles 3 made of gold with a diameter of 60nm are functionalised at first in analogy to the method used in FIG. 1.Herein, the shorter primer sequence 5 ID9 is used, as at the lowerannealing temperature and the higher salt concentrations a higherspecificity in the hybridisation with the original 12 can be achieved.The ratio of oligonucleotide 4 ID9 to oligonucleotide 4 ID2 amounts to1:9. After functionalisation and six washing steps, the firstnanoparticles 3 are present in a concentration of 200 pM in a PBS buffer(20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01% azide, 1 mM EDTA, pH 7.5).The amplification reaction is carried out in 10 μl PCR mixture in 100 μlsample tubes (1 μl 10× reaction buffer for the Klenow fragmentexo-(contains no dNTPs and no polymerase; fermentas), 0.2 μl Klenowfragment exo-(fermentas), 1 μl dNTPs, each 2.5 mM (fermentas), 0.2 μlNaCL 5M, 0.2 μl MgCl2 250 mM, 0.2 μl MgSO4 250 mM, 1 μl firstnanoparticles 3 in a concentration of 200 pM, 1 μl reversed primer IDG500 nM, 1 μl oligonucleotide ID3 as original 12 (herein, the original 12is present in the PCR mixture at a concentration of, e.g., 0 pM, 10 pMor 20 pM), 4.2 μl water). The quantities of salt used herein aresignificantly higher than in the example from FIG. 10.

With the DNA polymerase 10 from FIG. 10, no sufficient amplificationwould be possible in these salt concentrations. As shown in FIG. 7, thesample tubes 24 are temperature-controlled to 37° C. in a glass cuvette25 in a water bath 26. The temperature of 37° C. is, herein, annealingtemperature as well as elongation temperature. The laser 16 serves toexcite the first nanoparticles 3 and is a frequency-doubled diode-pumpedNd:YAg laser (coherent Verdi V10), which is focussed into the sampletubes 24 in the water bath 26 at an output power of 1.5 W with aF-theta-lens (Jenoptik, focal distance 100 mm) behind a mirror scanner18 (Cambridge Technologies, Pro Series 1). The mirror scanner 18 permitsto move the focus through the sample tubes 24 row by row, as alreadyshown in FIG. 3, and thus to involve the entire reaction volume 2 in theoptothermal amplification. Per sample tube 24, 1000 rows are scannedwith the focus at a distance of approximately 5 μm at a row velocity inthe sample tube 24 of approximately 5 m/s. This corresponds to one cyclein the first sample tube 24. Subsequently, all the other sample tubes 24are scanned one after the other such that each sample tube 24 hascompleted one cycle. After a waiting period of 40 s after the scanningof the first sample tube 24, the next cycle is started and this isrepeated until each sample tube 24 has completed 35 cycles in total.From left to right in FIG. 11, the first three sample tubes 24 havereceived the said PCR mixture including Klenow fragment 29 and dNTPs.The sample tubes 24 four till six contain Klenow fragment 29, but nodNTPs and the sample tubes 24 seven till nine contain dNTPs, but noKlenow fragment 29. The sample tubes 24 one, four and seven contain nooriginal 12, two, five and eight contain 10 pM original 12 and three,six and nine contain 20 pM original 12 as shown in the row below thediagram in FIG. 11. After all the sample tubes 24 have completed 35cycles, they are removed from the water bath 26. A test probe 21corresponding to the one from FIG. 9 is used in the analysis of theamplification reaction. For the hybridisation with the test probe 21, amodified phosphate buffer is used (13 mM PBS, 200 mM NaCl, 0.02% Tween20, 1 mM EDTA, 20 mM sodium citrate, 1 μg/ml PVP10, pH 7.5). 10 μlhybridisation solution contain 3.35 μl of the modified phosphate buffer,3.3 μl formamide, 0.6 μl NaCl 5M, 0.25 μl of the 200 pM test probesolution and 2.5 μl of the corresponding PCR solution after theamplification). The verification of the connection of firstnanoparticles 3 and test probes 21 is carried out by optothermalexcitation of the nanoparticles 8. FIG. 11 shows the transmissionchange, which is produced by the laser pulse and the resultantdehybridisation of DNA between the nanoparticles 8 and which is ameasure for the presence of gold-DNA-gold-bonds in the solution; theconcentration of the original 12 before the reaction is shown below thediagram.

On the left side of FIG. 11 in section A, the results for the sampletubes 24 one till three are shown, which contain the components Klenowfragment 29 and dNTPs required for the PCR. Here, it is evident thatwith an increasing amount of copies of the original 12, the transmissionchange measured as an indicator for the measure of gold-DNA-gold-bondsdecreases. The non-thermal-stable Klenow fragment 29 can carry out anamplification of the original 12 in this embodiment, even though it doesnot tolerate high temperatures. As in the present optothermalamplification reaction, the sample 11 is only heated locally, the Klenowfragment 29 experiences only little thermal strain and can amplify theoriginal 12 over many cycles without being destroyed. In FIG. 11 in themiddle in section B, the results of the sample tubes 24 four till six,which contain no dNTPs, are shown. Here, no significant transmissionchange is detectable in any of the concentrations of the original 12used. This means that no amplification has taken place. In section C inFIG. 11 on the right the results of the sample tubes 24 seven till nineis shown, which contain no Klenow fragment 29. Here, again, noamplification has taken place. This example shows that thenon-thermostable Klenow fragment 29 can also be used in performing anamplification reaction.

In the embodiment shown in FIG. 12, the optothermal amplification isshown depending on the movement of the laser beam through the reactionvolume 2. In this, concentrations of the original of 0, 1, 5, 20 and 50pM are chosen in the 100 μl sample tubes 24, as shown in the row belowthe diagram in FIG. 12. The sample tubes 24 are temperature-controlledto 60° C. in a glass cuvette 25 in a water bath 26 as shown in FIG. 7,wherein 60° C. is annealing temperature as well as elongationtemperature. The optothermal heating is created by the laser 16, whichconsists of a frequency-doubled diode-pumped Nd:YAg laser (coherentVerdi V10), which is focussed into the sample tubes 24 in the water bath26 at an output power of 3 W with a F-theta-lens (Jenoptik, focaldistance 100 mm) behind a mirror scanner 18 (Cambridge Technologies, ProSeries 1). FIG. 12 shows on the left side in section A five sample tubes24 with increasing concentrations of the original 12, which were eachexcited optothermally for one second, wherein the laser focus wasresting in the middle of the reaction volume 2 without movement. Afterthe first sample tube 24 was irradiated in this manner for one second,it is not irradiated for 40 s. This corresponds to one cycle in thefirst sample tube 24. During the waiting period of 40 s, the remainingsample tubes 24 complete the first cycle. The cycles are repeated 35times in total. The detection of the hybridisation between nanoparticles8, which contain primer sequences 5, and the test probes 21 is carriedout by means of optothermal excitation of nanoparticles 8 as alreadyshown in FIG. 10. As shown in FIG. 12 on the left side in section A,without movement of the laser focus, no influence of the concentrationof the original 12 on the transmission change can be observed. Thisshows, that no significant amplification of the original 12 has takenplace. The reason is that only a small fraction of the entire reactionvolume 2 is in focus and only the nanoparticles 8 in this partial volumetake part in the reaction. On the right side of FIG. 12 in section B,five sample tubes 24 are shown with increasing concentration of theoriginal 12 from left to right, which sample tubes 24 were each excitedfor one second, wherein additionally, the laser focus was moved throughthe reaction volume 2. In this way, as already shown in FIG. 3, theentire reaction volume 2 is scanned row by row and thus involved in theoptothermal reaction. In each sample tube 24, 1000 rows at a distance ofapproximately 5 μm are scanned with the focus at a row velocity in thesample tube 24 of approximately 5 m/s. This corresponds to one cycle inthe first sample tube 24. Subsequently, the next sample tubes 24 arescanned one after the other, until all five sample tubes 24 are scanned.After a waiting period of 40 s measured from the scanning of the firstsample tube 24, the next cycle is started and this is repeated 35 times.From FIG. 12 on the right in section B it is evident that withincreasing concentration of the original 12, the transmission changemeasured decreases. This is an indication for the decreasing measure ofgold-DNA-gold bonds. Only through the movement of the focus and at anunchanged laser power and duration of irradiation, amplification takesplace as the movement of the focus involves a large part of thenanoparticles 8 in the sample 11 in the amplification reaction.

In FIG. 13, an apparatus for performing the method according to theinvention is shown, wherein a light source 17 directs a light beamthrough an optional first objective 30 on a deflecting element 32, e.g.,a mirror, and through an optional second objective 31 onto a sample tube24. In this, the sample tube 24 is mounted on a rotatable unit 33together with further sample tubes 24 such that by turning the unit 33,different sample tubes 24 can be illuminated at different times. Thus,advantageously, it is achievable to excite a large number ofnanoparticles 8 present in the sample tubes 24, even with a light source17 with a low power.

FIG. 14 shows a section of a nanoparticle 8 according to the inventionwhich contains filling molecules 9 and oligonucleotides 4 on itssurface. Conventional DNA polymerases 10 synthesise a complementarystrand (dashed) along the oligonucleotides 4. During this, the large,conventional DNA polymerases 10 experience steric hindrance through thefilling molecules in FIG. 14a . In FIG. 14b , the DNA polymerases 10experience steric hindrance by neighbouring oligonucleotides 4. Thesteric hindrance in FIGS. 14a and 14b can each lead to premature strandbreak of the newly synthesised strand. The Klenow fragment 29 in FIG.14c can—as it is smaller than the conventional DNA polymerases 10—reachthrough between the filling molecules 9 and the oligonucleotides 4 andcan thus finish synthesising the new strand till the end. Even if theKlenow fragment 29 cannot reach through the filling molecules, theKlenow fragment 29 can still reach closer to the sterically hinderingfilling molecules 9 with its active centre, thus a potential strandbreak of the newly synthesised strand occurs only later. Hence, smallerpolymerases enable the more effective use of primer sequences close toparticle surfaces. Additionally, the locally produced heat can be usedparticularly effectively for the denaturing step in close proximity ofthe nanoparticle surface. In addition to this, a counter sequence 34with the sequence A′ is shown. The counter sequence 34 is complementaryto a oligonucleotide 4 with the sequence A on the nanoparticle 8 andserves to neutralise oligonucleotides 4 with the sequence A, whichunintentionally detach from the nanoparticles 8, such that theoligonucleotides 4 cannot act as free primers 7.

In the embodiment in FIG. 15, the optothermal amplification usingnanoparticle oligonucleotide-conjugates is shown, wherein the covalentbond between first nanoparticles 3 and primer sequences 5 is carried outwith two thiols. To this end, first nanoparticles 3 made of gold with adiameter of 60 nm are functionalised with oligonucleotides 4 (accordingto J. Hurst, supra). In this, oligonucleotides 4 ID10 (IDT Technologies,Inc.) are used, which compared to oligonucleotide ID1 carry a dithiolinstead of a thiol on their 5′-end. After functionalisation and sixwashing steps, the first nanoparticles 3 are present at a concentrationof 200 pM in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01%,azide 1 mM EDTA, pH 7.5). The amplification reaction is performed in atotal volume of 10 μl in 200 μl sample tubes 24 (5 μl DreamTaq PCRMastermix 2× (fermentas), 0.1 μl NaCl 5M, 0.1 μl MgCl₂ 250 mM, 0.1 μlMgSO₄ 250 mM, 1 μl of the functionalised first nanoparticles 200 pM, 1μl reversed primer IDG 500 nM, 1 μl oligonucleotide 4 ID3 (as original12 to be amplified) dissolved at a concentration of 0 or 200 pM,respectively, in water with 100 nM oligonucleotide 4 ID4 (herein,oligonucleotide 4 ID4 serves to saturate surfaces, e.g., during thestorage of the original 12 before the reaction), 1.7 μl water). As shownin FIG. 7, the sample tubes 24 are temperature controlled to 60° C. in aglass cuvette 25 in a water bath 26, which is annealing temperature aswell as elongation temperature. The first two sample tubes 24 areinserted into the water bath as negative controls, but are not hit bythe laser beam. Initial concentrations of the original 12 are chosen tobe 0 pM in the first sample tube 24 and 20 pM in the second sample tube24. The third and fourth sample tube is treated optothermally. Theoptothermal heating is performed by the laser 16, which consists of afrequency-doubled diode-pumped Nd:YAg laser (Coherent Verdi V10), whichis focussed into the sample tubes 24 in the water bath 26 at an outputpower of 3 W with a F-theta-lens (Jenoptik, focal distance 100 mm)behind a mirror scanner 18 (Cambridge Technologies, Pro Series 1). Foreach sample tube 24, 500 rows are scanned with a distance ofapproximately 10 μm with the focus at a row velocity in the sample tubeof approximately 5 m/s. This corresponds to one cycle in the thirdsample tube 24. Subsequently, the fourth sample tube 24 is scanned suchthat third and fourth sample tube have completed one cycle. After awaiting period of 40 s after the scanning of the third sample tube 24,the next cycle is started and this is repeated until third and fourthsample tube 24 have each completed 35 cycles. As initial concentrationof the original 12, 0 pM is chosen for the third sample tube 24 and 20pM is chosen for the fourth sample tube 24. After the third and fourthsample tube have completed 35 cycles, all four sample tubes 24 areremoved from the water bath 26. A test probe 21 serves to examine theeffect of the laser cycles and the concentration of the original 12,which test probe 21 is preferably able to hybridise under the primer andhybridisation conditions chosen to the first nanoparticles 3functionalised with primer sequences 5, which first nanoparticles 3 arenot blocked by complementary copies of the original 12, which wereproduced in the amplification reaction. This corresponds to the testprobe 21 as shown in FIG. 9. The production of the test probe 21 and thehybridisation conditions were already described in FIG. 10. The diagramsin FIG. 15 show absorbance spectra of the hybridisation solution, whichcontains the test probe 21 as well as the corresponding PCR solutionfrom the optothermal amplification reaction, which contains the firstnanoparticles 3. The absorbance spectra were recorded in a quartzcuvette with a 3 mm optical path in a Varian Cary 50 spectrometer. Inthe diagrams the solid line shows the absorbance spectrum immediatelyafter mixing the PCR solution and the optothermal amplificationreaction, which contains the first nanoparticles 3 with the test probe21; the dashed line is recorded 6 minutes after hybridisation and thedotted line after 12 minutes hybridisation. In FIG. 15a , shown are thespectra during the hybridisation of the test probe 21 with thenanoparticles 8 of the PCR product from the first sample tube, whichcontained no original 12 before the amplification reaction and whichexperienced no optothermal treatment. A clear red shift and broadeningof the plasmon resonance of the nanoparticles 3 is seen with increasinghybridisation time as a hybridisation between test probes 21 and primersequences 5 takes place on the first nanoparticles 3. A comparablehybridisation can also be seen in FIG. 15b , which shows thehybridisation of the test probe 21 with nanoparticles 8 of the PCRproduct from the second sample tube, which contained 20 pM original 12before the amplification reaction and has received no optothermaltreatment. A comparable hybridisation can also be seen in FIG. 15c ,which shows the hybridisation of the test probe 21 with thenanoparticles 8 of the PCR product from the third sample tube, whichcontained no original 12 before the amplification reaction, but receiveda optothermal treatment. The hybridisation in FIG. 15c shows that primersequences 5 are still bound to the first nanoparticles 3 afteroptothermal treatment. Only in FIG. 15d , which shows the hybridisationof the test probe 21 with nanoparticles 8 of the PCR product from thefourth sample tube, which contained 20 pM original 12 before theamplification reaction and received an optothermal treatment, almost nochange of the absorbance spectra is seen with increasing hybridisationtime. Only in the latter case, a sufficient number of copies of theoriginal 12 were produced during the amplification reaction, whichcopies now block primer sequences 5 on the first nanoparticles 3 andthus prevent a hybridisation with the test probes 21. This example showsthat the optothermal amplification reaction also functions if primersequences 5 are bound covalently to dithiols on the surface of the firstnanoparticles 3 and that absorbance spectra for the detection of theconcentration of the copies of the original 12 can be used after theamplification reaction.

The features disclosed in the present description, the claims and thedrawings can be of relevance individually as well as in any combinationfor the realisation of the invention in its various embodiments.

REFERENCE NUMBER LIST

-   1 nucleic acid-   2 reaction volume-   3 first nanoparticles-   4 oligonucleotide-   5 primer sequence-   6 spacer sequence-   7 primer-   8 nanoparticle-   9 filling molecule-   10 DNA polymerase-   11 sample-   12 original-   13 complement-   14 forward primer-   15 reverse primer-   16 laser-   17 light source-   18 mirror scanner-   19 mirror-   20 first oligonucleotide-   21 test probe-   22 second nanoparticle-   23 second oligonucleotide-   24 sample tube-   25 glass cuvette-   26 water bath-   27 first laser-   28 second laser-   29 Klenow fragment-   30 first objective-   31 second objective-   32 deflecting element-   33 rotatable unit-   34 counter sequence-   35 photo diode

What is claimed is:
 1. A method for the amplification of nucleic acids,wherein nanoparticles in a sample in a reaction volume transfer heat totheir environment through excitation, wherein the nanoparticles areexcited through electromagnetic waves, wherein the method comprises amovement of the sample relative to an exciting field, wherein themovement takes place such that at different times, nanoparticles indifferent partial volumes of the sample are excited.
 2. The methodaccording to claim 1, wherein through the excitation of thenanoparticles, the environment of the nanoparticles is heated locally.3. The method according to claim 1, wherein the nucleic acids areamplified by a polymerase chain reaction.
 4. The method according toclaim 1, wherein the nanoparticles are excited by a laser.
 5. The methodaccording to claim 1, wherein the nanoparticles are conjugated witholigonucleotides.
 6. The method according to claim 5, wherein one kindof conjugates of the nanoparticles and the oligonucleotides isconjugated with forward primers as well as reverse primers.
 7. Themethod according to claim 5, wherein filling molecules are attached tothe nanoparticles.
 8. The method according to claim 5, wherein theoligonucleotides on the nanoparticles contain a spacer sequence as apartial sequence.
 9. The method according to claim 5, wherein the heat,which is transferred to the environment of the nanoparticles by theexcitation of the nanoparticles, is sufficient to dehybridise theoligonucleotides on the surface of the nanoparticles from nucleic acidshybridised with the oligonucleotides.
 10. The method according to claim1, wherein the method further comprises a global heating step.
 11. Themethod according to claim 1, wherein the method further comprises anannealing step and the annealing temperature is equal to an elongationtemperature.
 12. The method according to claim 1, wherein at any onetime during the method only a part of the nanoparticles is heated byexcitation.
 13. The method according to claim 1, wherein the methodfurther comprises use of a thermolabile DNA polymerase.
 14. The methodaccording to claim 1, wherein the method further comprises concentrationof the amplification product and the concentration of the product of theamplification reaction is determined using test probes.
 15. A setup,wherein nanoparticles in a sample in a reaction volume transfer heat totheir environment through excitation, wherein the nanoparticles areexcited through electromagnetic waves, wherein in the setup, a movementof the sample relative to the exciting field takes place such that atdifferent times, nanoparticles in different partial volumes of thesample are excited.
 16. The setup according to claim 15, wherein thesetup comprises the reaction volume and a source for emitting theexciting field, and wherein the movement takes place by at least one ofthe following: the source is configured for moving to induce a movementof the sample relative to the exciting field; the reaction volume or thesample are configured for moving to induce a movement of the samplerelative to the exciting field; the setup comprises an elementconfigured for directing the exciting field in different partial volumesof the reaction volume to induce a movement of the sample relative tothe exciting field.
 17. The setup according to claim 15, wherein thesetup is suitable for amplifying nucleic acids in the sample by apolymerase chain reaction.